This invention is concerned with a dry diagnostic reagent of improved sensitivity and susceptibility to quantification for use in the fluorescent method for the detection of antinuclear factor (ANF) in the diagnosis of systemic lupus erythematosus (SLE) and other autoimmune diseases composed of specially prepared, fixed and stabilized fetal calf thymocytes.
The fluorescent antibody technique for the detection of antinuclear factor in the diagnosis of systemic lupus erythematosus and other autoimmune diseases using nuclear cell material, including calf thymus nuclei, is known. Friou, G. J., et al., Journal of Immunology, Vol. 80, 324-329 (1958); Widelock, D., et al., Am. J. Pub. Health, 51, 829-835 (1961); Keffer, J. H., Proc. Ann. Meeting, College Am. Path. and Ann. Soc. Clin. Path., No. 16, pg. 87 (1970); and Burrows, S., et al., Jour. Med. Soc. N.J., Vol. 68, pgs. 647-649 (1971).
Friou et al., supra, have demonstrated that the fluorescent antibody technic can be utilized to illustrate the union of lupus erythematosus (LE) serum factor with nuclear material. Of special interest is Friou et al's., demonstration of the usefulness of the fluorescent antibody technic in the diagnosis of SLE. Friou et al., reported that calf thymus nuclei were satisfactory for the detection of the SLE factor and concluded that the most satisfactory preparation for testing human sera for SLE factor was calf thymus nuclei.
In Friou et al., a drop of a dilute suspension of calf thymus nuclei was allowed to dry on a gelatin-coated slide. The slide was then flooded with the serum being tested and allowed to stand in a moist tray for 30 minutes. It was then washed with isotonic buffered saline, pH 7.0, for 10 minutes. Finally the material was exposed to slightly diluted fluorescent conjugate for 30 minutes, washed again, mounted in buffered glycerol under a cover slip and examined.
Widelock et al., supra, also report on work with a fluorescent antibody procedure for lupus erythematosus using calf thymus cells. Widelock et al. conclude that screening for systemic lupus erythematosus using the fluorescent antibody technique with calf thymus nuclei was more sensitive than the performance of other tests and that diseases other than SLE were found to be positive by such technique. Widelock et al. report that the use of calf thymus nuclei necessitated obtaining a fresh calf thymus gland which should be frozen at -40.degree. C. within four hours after death of the animal and that decreasing sensitivity of the test was observed as the calf thymus aged. The fluorescent technic of Widelock et al., utilizing calf thymus cells, was based on the method described by Friou et al., supra. The Widelock et al. report is essentially a confirmation of the previous report by Friou et al., supra.
In Proc. Annual Meeting, College of Am. Path. and Ann. Soc. Clin. Path., Sept. 11-19, 1970, page 87, Keffer, J. H., notes the value of fluorescent antinuclear antibody testing as an objective replacement for the "L.E. Cell Test" and emphasizes the simplicity of fluorescent microscopy. Although not so reported in the aforementioned Keffer, J. H. publication, Keffer, J. H. has used fetal calf thymus as a source of thymocytes in fluorescent antinuclear antibody testing. However, Keffer, J. H.'s known work did not involve a dry diagnostic reagent composed of specially prepared, fixed and stabilized fetal calf thymocytes on a microscope slide ready for use in an immunofluorescent test for the detection of human antinuclear factor in human serum or plasma as set forth herein.
Barnes, R., et al., in Ann. rheum. Dis. 21:287-291 (1962) report on a comparison between a commercial preparation of nucleoprotein coated latex particles and the fluorescent method for the detection of serum antinuclear factor and conclude that the nucleoprotein coated latex particle does not appear to be a satisfactory substitute for the immunofluorescent technique. Barnes, R., et al., appear to have used human or bovine thyroid as a source of thymocytes.
The combination of features which distinguish the present invention from the above publications include: the use of fetal calf thymus as a source of thymoctyes; sieving to isolate individual cells; preparing smears of thymocytes on microscope slides by air-drying and chemical fixation; and fixed and stabilized thymocyte slides ready for use in an air-tight container with dessicant. The diagnostic reagent of the present invention is fixed (dried chemically) on the slide ready for instant use in the detection of antinuclear factor and the diagnosis of SLE and other autoimmune diseases. The reference products require slide preparation.
The diagnostic reagent of the present invention can be utilized by hospitals, clinical laboratories and by physicians as a diagnostic reagent for use in the fluorescent antinuclear factor test for the detection and quantitation of serum factors associated with autoimmune diseases such as systemic lupus erythematosus (SLE). Other "autoimmune diseases" which may be diagnosed by this reagent include connective tissue diseases, rheumatoid arthritis, szleroderma and dermatomyositis. The invention disclosed herein provides a reagent for the lupus erythematosus fluorescent ANF test that is convenient, ready to use, stable, highly sensitive and lends itself to standardization.